Title

Author

Document Type

Dissertation

Date of Degree

Spring 2016

Degree Name

PhD (Doctor of Philosophy)

Degree In

Genetics

First Advisor

Richard J.H. Smith

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a rare renal disorder characterized by thrombotic microangiopathy, thrombocytopenia, and acute kidney injury. Its pathogenesis has been attributed to a ‘triggering' event that leads to dysregulation of the complement cascade at the level of the endothelial cell surface. Consistent with this understanding of the disease, mutations in complement genes have been definitively implicated in aHUS. However, the existence of other genetic contributors is supported by two observations. First, in ~50% of cases, disease-causing variants are not identified in complement genes, and second, disease penetrance is typically incomplete and highly variable.

To test this hypothesis, we identified pathways established to have crosstalk with the complement cascade, focusing initially on the coagulation pathway. Using targeted genomic enrichment and massively parallel sequencing we screened 36 European-American patients with sporadic aHUS patients for genetic variants in 85 complement and coagulation genes, identifying deleterious rare variants in several coagulation genes. The most frequently mutated coagulation gene in our study cohort was PLG, which encodes a zymogen of plasmin and plays key role in fibrinolysis. These results implicate the coagulation pathway in the pathogenesis of aHUS.

Based on this outcome, we developed a clinical genetic testing panel to screen disease-related genes in a group of ultra-rare complement-mediated diseases that includes, in addition to aHUS, thrombotic thrombocytopenic purpura (TTP), C3 glomerulonephritis (C3GN) and dense deposit disease (DDD) patients. Data from 193 patients validate the usage of this panel in clinical practice and also provide confirmatory insight into the pathogeneses of these diseases. Specifically, we found that in aHUS and TTP patients, variants were frequently identified in complement regulator genes, while in C3GN and DDD patients, variants were additionally found in C3 convertase genes.

To understand variability in disease penetrance, we completed targeted genetic screening in two aHUS families grossly discordant for disease penetrance, identifying in one family a co-segregating Factor X-deficiency variant (F10 p.Glu142Lys) that abrogated the effect of the complement mutation. Functional studies of the F10 p.Glu142Lys variant show that it decreases Factor X activity predicting to a hypo-coagulable state and further illustrating the importance of complement-coagulation crosstalk in exacerbating, but also mitigating the aHUS phenotype.

In our final studies, we have sought to complete a comprehensive analysis for other potentially related pathways by using bioinformatics to identify candidate pathways coupled with whole exome sequencing. Preliminary data from 43 aHUS patients and 300 controls suggest that pathways for dermatan and heparan sulfate synthesis, which are relevant to the formation of the extra-cellular matrix and cell surface adhesion, may be implicated in the aHUS.

Public Abstract

Atypical hemolytic uremic syndrome (aHUS) is a rare but life-threating disease featured by abnormal blood clots forming in small vessels which causes red blood cells break down, reduced level of platelets, and kidney failure. Previous studies have linked the disease to pathogenic mutations in genes of the complement pathway, which is part of the immune system. However, other genes may also link to aHUS since previous genetic tests were too narrow and many patients do not carry disease-related mutation in known aHUS genes. Therefore, we comprehensively screened genes in the complement pathway, and that are responsible for blood clots formation and removal (the coagulation pathway). We found that mutations in PLG, which repress the activity of blood clots removal, associate with the occurrence of aHUS. In addition, a mutation in F10 gene, which represses the blood clots formation, can cancel the effect of a well-known aHUS mutation in CFH gene. With these evidences, we were able to first implicate the coagulation pathway into the disease mechanism. Next, in order to provide better patients care, we translated our research results and techniques to build an advanced clinical genetic testing panel that provides accurate and informative results, and significantly reduces the cost and waiting time. Finally, we expanded the research scope and tested all genes in human genome. Preliminary results show that genes responsible for forming the protective layer above the surface of vessel wall (endothelial glycocalyx) may also link to aHUS.